One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation, orchestrated by enzymes known as kinases, represents one course by which intracellular signals are propagated from molecule to molecule resulting in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases, which remove phosphate moieties. It is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
One major kinase signal transduction pathway involves the enzyme known as protein kinase C. Protein kinase C (PKC) is a family of enzymes that are physiologically activated by 1,2-diacylglycerol (DAG) and other lipids. When activated, the isozymes bind to membrane phospholipids or to membrane receptors and anchor the enzymes in a subcellular compartment reviewed in (Liu and Heckman, Cell. Signal., 1998, 10, 529-542).
Protein kinase C isozymes differ in number and expression level in different cell lines and tissues. To date, 11 different isozymes (alpha, betaI, betaII, gamma, delta, epsilon, nu, lambda, mu, theta and zeta) have been identified and they have been divided into three groups based on their differential expression patterns and cofactor requirements. Interest in protein kinase C as a therapeutic target was generated by the finding that it is the major cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells (Liu and Heckman, Cell. Signal., 1998, 10, 529-542).
Protein kinase C-theta (also known as PKC-.theta., PKCT, PRKCT, nPKC-theta and PRKCQ), one of the novel serine/threonine protein kinase C isoforms (nPKC), is expressed ubiquitously in tissues with the highest levels found in hematopoietic cell lines, including T-cells and thymocytes. (Baier et al., J. Biol. Chem., 1993, 268, 4997-5004; Keenan et al., Immunology, 1997, 90, 557-563; Meller et al., Cell. Immunol., 1999, 193, 185-193; Wang et al., Biochem. Biophys. Res. Commun., 1993, 191, 240-246). This isozyme has been shown to function in a calcium-independent fashion, and transient overexpression of the protein in murine thymoma cells resulted in transcriptional activation of an interleukin-2 promoter-driven construct (Baier et al., Eur. J. Biochem., 1994, 225, 195-203), indicating a role for protein kinase C-theta in T-cell signaling pathways.
Subsequent characterization of the role of protein kinase C-theta in T-cell systems has shown that it is also involved in cell cycle control (Resnick et al., J. Biol. Chem., 1998, 273, 27654-27661), cellular activation (Monks et al., Nature, 1997, 385, 83-86), AP1 transcription factor stimulation (Baier-Bitterlich et al., Mol. Cell. Biol., 1996, 16, 1842-1850), and the etiology of AIDS (Smith et al., J. Biol. Chem., 1996, 271, 16753-16757). The human protein kinase C-theta gene has been localized to chromosome 10p15. This finding is noteworthy since deletions and translocations in this chromosomal region have been associated with several human T-cell disorders (Erdel et al., Genomics, 1995, 25, 595-597; Kofler et al., Mol. Gen. Genet., 1998, 259, 398-403). Methods to modulate the immune response, particularly the activity and differentiation of T-cells, are disclosed in U.S. Pat. No. 5,776,716. These methods involve the use of peptides to block interactions of protein kinase C-theta with fyn, a receptor for the activated protein kinase C-theta enzyme (Ron et al., 1998).
Protein kinase C-theta has been shown to be an upstream activator of the c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. This pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation (Moriguchi et al., Adv. Pharmacol., 1996, 36, 121-137). Expression of a constitutively active form of protein kinase C-theta resulted in strong activation of the JNK/SAPK pathway in Jurkat T-cells, while a dominant negative form interfered with activation of interleukin-2 promoter transcription (Ghaffari-Tabrizi et al., Eur. J. Immunol., 1999, 29, 132-142). This pathway has been further characterized and shown to be activated only in conjunction with a calcium signal delivered through the protein calcineurin (Avraham et al., Eur. J. Immunol., 1998, 28, 2320-2330).
Protein kinase C-theta has also been implicated in other cellular processes including apoptosis (Datta et al., J. Biol. Chem., 1997, 272, 20317-20320), cytoskeletal arrangement (Pietromonaco et al., J. Biol. Chem., 1998, 273, 7594-7603; Simons et al., Biochem. Biophys. Res. Commun., 1998, 253, 561-565), proliferation (Passalacqua et al., Biochem. J., 1999, 337, 113-118), and angiogenesis and wound repair (Tang et al., J. Biol. Chem., 1997, 272, 28704-28711). In rat models, protein kinase C-theta has been shown to be involved in insulin signaling (Griffin et al., Diabetes, 1999, 48, 1270-1274) and it is currently believed to play a role in the development of diabetes in humans (Kellerer et al., Diabetologia, 1998, 41, 833-838). The pharmacological modulation of protein kinase C-theta expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of protein kinase C-theta and investigative strategies aimed at modulating the PKC family of isozymes have involved the use of antisense oligonucleotides and PKC inhibitors such as staurosporine, which generally and nonspecifically inhibits PKC isoforms. Combination treatments of the two are disclosed in the PCT publication WO 98/07415 and WO 97/32589. However, the antisense compounds listed only target protein kinase C-alpha mRNA (Muller et al., 1997; Prescott, 1998).
Antisense mediated inhibition of PKC isoforms is also disclosed in U.S. Pat. Nos. 5,703,054 and 5,885,970 (Bennett and Dean, 1999; Bennett and Dean, 1997) as well as the PCT publication WO 95/02069 and DE 19740384A1 (Bennett et al., 1995; Haller, 1999). Oligomers comprised of subunits, of which one subunit is a protein nucleic acid, targeting protein kinase C isoforms are disclosed in the PCT publication WO 95/03833 (Dean, 1995). However these studies involve inhibition of specific isoforms and do not include the protein kinase C-theta isozyme. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting protein kinase C-theta function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of protein kinase C-theta expression.
The present invention provides compositions and methods for modulating protein kinase C-theta expression.